One impediment to gonadotropin hormone replacement therapy has been the need for frequent repeated injections in order to maintain efficacious serum levels of the hormone. For example, current pharmacological formulations of follicle stimulating hormone (FSH) must be administered daily, often for 8 to 12 days when used to induce ovulation (LeContonnec et al, 1994). This dosing regimen is required due to the relatively short serum half-life of FSH. Thus, the development of more stable, longer-acting FSH formulations has been an area of active research.
Glycoprotein hormones, including FSH, luteinizing hormone (LH), thyrotropin stimulating hormone (TSH) and chorionic gonadotropin (CG), are heterodimers comprised of two non-covalently bound subunits, α and β (Pierce et al, 1981). While the α-subunit is interchangeable among the hormones of this family, the β-subunit is unique and primarily responsible for the biological specificity of hormone action (see FIGS. 17 and 18 for the sequences of the hFSH β and α subunits, respectively).
The β subunits of hCG and hLH share greater than 85% sequence identity through the N-terminal 112 amino acids (Pierce et al, 1981). In addition, these two hormones share a common receptor and elicit similar biological activity following receptor binding. However, the serum half-life of hCG is almost five times that of hLH (Porchet et al, 1995; Saal et al, 1991; Yen et al, 1968). The main structural difference between β-hCG and β-hLH is an additional carboxy terminal peptide sequence on the β-hCG subunit, corresponding to amino acids 113–145 (Matzuk et al, 1990). This carboxy-terminal peptide, or “CTP”, is now known to be the key determinant of the decreased metabolism and excretion of hCG, and thus of its long serum half-life, the longest among the glycoprotein hormones. The biological role of the carboxy-terminal extension of hCG is discussed in Matzuk et al, (1990). Surprisingly, the sequence of the CTP peptide itself is not responsible for the stability of this protein. Rather, it is the presence of four O-linked sugars that confer its remarkable stability. The characterization of the O-glycosylation sites of the hCG β subunit is described in Sugahara et al, (1996).
Many integral membrane proteins and secretory proteins are glycosylated. The most common type of glycosylation, called “N-linked”, occurs when a sugar moiety is linked to the amide nitrogen of an asparagine residue within the consensus sequence Asn-X-Ser or Asn-X-Thr. The carbohydrate moieties of the gonadotropin hormones play an important role in the hormone's functionality. For example, appropriate glycosylation is required for protein folding and hormonal signal transduction. The importance of the asparagine-linked oligosaccharides of the hCG β subunit in correct disulfide bond pairing and thus on assembly and secretion are discussed in Feng et al, (1995) and Matzuk et al, (1990).
The O-linked glycosylations of the CTP sequence of hCG are so-named because the sugar moiety is linked to the hydroxyl group of either serine or threonine. This kind of linkage is much less common and, in contrast to N-linked moieties, O-linked sugars are not required for the hormonal activity of hCG. Schematic examples of N-linked and O-linked carbohydrates are shown in FIG. 16.
The importance of the CTP in promoting hormone stability was demonstrated by the construction of a fusion protein consisting of the CTP portion of the β-subunit of hCG and the carboxy terminus of β-hFSH (Fares et al, 1992). This β-hFSH-CTP fusion produced a long-acting hFSH agonist which was able to dimerize with a coexpressed α-subunit to produce a functional FSH hormone. Importantly, this β-hFSH-CTP demonstrated similar in vitro bioactivity and substantially increased in vivo bioactivity compared with preparations of native hFSH.
Thus, merely adding the CTP sequence to β-hFSH was sufficient to increase the biological activity of the hormone, most likely through an increase in serum-half life. Indeed, recent pharmacokinetic parameter estimates in humans have demonstrated that this β-hFSH-CTP analog has an elimination half-life 2 to 3 times longer than that of native recombinant hFSH (Bouloux et al, 2001).
While the administration of pharmacological preparations of hFSH alone has been used in the treatment of infertility, this approach is likely to be enhanced substantially by methodologies which simultaneously induce follicular vascularization.
The primary mediator of follicular vascularization is VEGF, which is induced in response to FSH stimulation (Dissen et al, 1994). Reproductive aging is associated with diminished follicular vascularity, resulting somewhat paradoxically in an increase of VEGF, presumably in response to local hypoxia (Van Blerkom et al, 1997). However, this adaptive response is often inadequate to reestablish appropriate vascular supply to the area.
Notably, improved vascularization has clinically been associated with improved oocyte yield, embryo morphology and pregnancy rates (Chui et al, 1997; Nargund et al, 1996). This is because perifollicular blood vessel development is critical to the health and integrity of the growing follicle.
Pharmacologic doses of exogenous VEGF can significantly ameliorate perifollicular hypoxia and improve follicular development in some patients. However, attempts to provide exogenous VEGF have suffered from severe side effects associated with the systemic administration of this growth factor.